Techniques have been developed to systematically identify and characterize herpes simplex virus genes that control pathogenic phenotypes displayed in experimental animal models of human disease. A series of viral variants that display altered pathogenic properties have been identified and these include: an HSV-1/HSV- 2 intertypic recombinant (RE6) which is completely and specifically non-neurovirulent upon intracranial inoculation of mice; a revertant of a temperature sensitive mutant that is not temperature sensitive in cultured cells but displays a temperature sensitive reactivation phenotype; a wild type strain of HSV-1 (KOS) that is non-neuroinvasive following peripheral inoculation of mice. These variants have been restored to wild type pathogenicity by recombination with libraries of cloned DNA fragments from pathogenic strains followed by selection of rescued recombinant viruses in the appropriate mouse tissue in vivo. A combined molecular and biological approach is described to: 1) determine the genes involved; 2) define the nature of the mutations on a nucleotide and/or amino acid level; 3) begin an analysis of the expression and in vivo function of the relevant genes at a tissue specific and single cell level. Similar experiments are described to determine the genetic basis for the generally increased virulence of HSV type-2 strains over type-1 strains in the mouse peripheral nervous system.